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    Factors inducing regeneration response in oat (Avena sativa L.) anther culture

    Marzena Warchoł1 & Ilona Czyczyło-Mysza1 & Izabela Marcińska1 & Kinga Dziurka1 & Angelika Noga1 & Kamila Kapłoniak1 & Marta Pilipowicz1 & Edyta Skrzypek1

    Received: 24 October 2018 /Accepted: 12 April 2019 / Editor: Yong Eui Choi # The Author(s) 2019

    Abstract The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the inductionmedium supplementedwith different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm3 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm3 kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

    Keywords Oat . Androgenesis . Pretreatment . Embryo-like structures . Plant growth regulators


    In vitro production of doubled haploid (DH) plants through androgenesis induction is a promising and convenient al- ternative to traditionally used techniques for rapid produc- tion of fully homozygous plants for breeding programs, marker identification, and gene mapping. A significant ad- vantage is that the system not only speeds up the process of obtaining homozygosity but also increases selection effi- ciency (Islam and Tuteja 2012). Androgenesis is defined as a developmental pathway, alternative to zygotic embryo- genesis, driven by a shift of the normal gametophytic

    development of microspore into the sporophytic develop- ment in which embryos and plants are inherited with the genetic traits from the male donor plant. There are two basic methods of androgenesis: anther and isolated micro- spore cultures. The former involves culturing whole an- thers, whereas in the latter, microspores are isolated from anthers prior to in vitro culture (Khound et al. 2013).

    In recent years, androgenesis-based methods have been particularly successful in Solanaceae, Brassicaceae, and Gramineae. Nevertheless, only a few species, such as barley (Hordeum vulgare L.), rape (Brassica napus L.), tobacco (Nicotiana spp.), wheat (Triticum aestivum L.), pepper (Capsicum annum), and rice (Oryza sativa), found application in breeding programs due to their high regenerative capacity and were considered an ideal source of information in micro- spore embryogenesis research (Forster et al. 2007). There are many reviews explaining the production and application of DH plants through androgenesis (Maluszynski et al. 2003; Forster et al. 2007; Germanà 2011). Nevertheless, scientifical- ly or economically important species, woody plants or Leguminosae species, still remain resistant to androgenesis induction (Skrzypek et al. 2008; Germanà 2009).

    Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11627-019-09987-1) contains supplementary material, which is available to authorized users.

    * Edyta Skrzypek e.skrzypek@ifr-pan.edu.pl

    1 Polish Academy of Sciences, Department of Biotechnology, The Franciszek Górski Institute of Plant Physiology, Niezapominajek 21, 30-239 Krakow, Poland

    In Vitro Cellular & Developmental Biology - Plant https://doi.org/10.1007/s11627-019-09987-1

    (2019) 55:595–604

    /Published online: 20 May 2019

    http://crossmark.crossref.org/dialog/?doi=10.1007/s11627-019-09987-1&domain=pdf http://orcid.org/0000-0003-3185-495X https://doi.org/10.1007/s11627-019-09987-1 mailto:e.skrzypek@ifr-pan.edu.pl

  • Haploid plants (HPs) of oat (Avena sativa L.) can be pro- duced by hybridizations either with maize (Sidhu et al. 2006; Marcińska et al. 2013; Nowakowska et al. 2015; Warchoł et al. 2016) or with pearl millet (Ishii et al. 2013) and by androgenesis in microspore/anther culture (Rines et al. 1997; Kiviharju et al. 2005; Kiviharju et al. 2017). Pioneering work on oat androgenesis was carried out by Rines (1983), who obtained the first three plants (one haploid and two diploids) from 65,000 anthers of the cultivar ‘Stout’. In the 1990s, suc- cessful plant recovery from oat anthers was reported by Sun et al. (1991), who recovered 12 green oat plants, including two haploids and one euploid. Kiviharju and Pehu (1998) reported many anther-derived embryos without successful plant regeneration. Later, Kiviharju et al. (2005) improved the anther culture method by several adjustments to older methods and reported up to 30 green plants per 100 anthers cultured for an individual cross. Recently, Ślusarkiewicz- Jarzina and Ponitka (2007) have described plants derived from anther culture from Polish oat cultivars.

    Androgenesis, like other haploid-inducing techniques, is influenced by several endogenous and exogenous factors. Genotype, physiological state, and growth conditions of donor plants, stage of male gametes, pretreatment of flower buds or anthers, in vitro culture medium composition, and physical factors during tissue culture together with their interactions are the main factors determining the androgenetic response in in vitro culture (Islam and Tuteja 2012; Murovec and Bohanec 2012; Ferrie et al. 2014). The application of suitable physiochemical factors promotes stress response that arrests microspores or young pollen grains in their gametophytic pathway. Temperature pretreatment, sucrose and nitrogen star- vation, and osmotic stress are the most commonly used trig- gering factors. Depending on the plant species and genotype, temperature stress can be applied by subjecting excised flower buds, whole inflorescences, or excised anthers to low (barley, wheat, maize, rice, triticale, rye) or high (Brassica species, tobacco, wheat) temperatures for several hours or days (Maluszynski et al. 2003; Maraschin et al. 2005). Ślusarkiewicz-Jarzina and Ponitka (2007) reported harvesting and cold-treating oat tillers at 4°C for a few days in N6mineral salt medium (Chu et al. 1975) with 2.0 mg/dm3 2,4-D. Sidhu and Davies (2009) also used 4°C temperature for oat andro- genesis induction for 6–9 wk. Temperature pretreatment is usually conducted at low temperatures, but sometimes, heat shock conditions of 32°C for hours or days are used in oat anther cultures (Kiviharju et al. 1998). Anthers were also heat shocked at 35°C for 24 h immediately after placing them on induction media (Rines 1983).

    The aim of the experiments was the induction of androgen- esis in anther culture of oat. The effects of cultivar, pretreat- ment, induction medium, and distance from the base of the flag leaf to the penultimate leaf of the panicle on the andro- genesis efficiency were tested.

    Materials and methods

    Oat (Avena sativa L.) cultivars derived from Strzelce Plant Breeding Ltd., PBAI Group, Strzelce, Łódź Voivodeship, Poland were used as sources of anthers for studying andro- genic abilities. Seeds of each genotype were sown singly into a mixture of soil with sand (3:1 v/v) in 3 dm3 pots. Donor plants were grown under controlled conditions at 21/17°C day/night, 16-h photoperiod, in a greenhouse under natural (solar) light during the day and sodium lamps (400 W, Philips SON-T AGRO, Philips Lighting, Eidhoven, the Netherlands) between 6 and 8 a.m. and additionally between 6 and 10 p.m. on cloudy days. Plants were fertilized with a liquid medium once a w (Hoagland and Arnon 1938).

    In the first experiment, 175 tillers of ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’ were cut when the panicle was enclosed within the leaf sheath (Fig. 1a). At this time, the majority of microspores were at the late uninucleate (Fig. 1b) to early binucleate stage (Fig. 1c). To correlate the stage of microspore development with tiller morphology, microspores were ob- served under a light microscope (SMZ 1500, Nikon, Tokyo, Japan) and the distance from the base of the flag leaf to the penultimate leaf of the panicle was measured and four dis- tance classes were designated: (A) 0.0–4.0 cm, (B) 4.1–8.0, (C) 8.1–12.0, and (D) 12.1–16.0.

    Oat tillers were covered with aluminum bags (Fig. 1d), dipped in Hoagland and Arnon (1938) liquid medium for 2 and 3 wk at 4°C. Next panicles were disinfected in 70% (v/v) ethanol (1 min), then in a 2.5% (w/v) solution of calcium hypochlorite (65% Ca(OCl)2 commercial product, Sigma- Aldrich®, Darmstadt, Germany) (7 min), and subsequently washed three times with sterile water. For embryo-like struc- tures (ELS) induction, anthers were aseptically isolated on C17 medium (Wang and Hu 1984) with the addition of 4- amino-3,5,6-trichloropicolinic acid (picloram), 2-methoxy- 3,6-dichlorobenzoic acid (dicamba), and kinetin. All growth regulators were added to the medium at a co